Microorganisms for waste treatment

ABSTRACT

The invention relates to treatment of waste with one or more microorganisms for the purposes of, including but not limited to, degrading waste, bioremediation of waste, enhancing waste stabilization, reducing contaminants in waste, reducing odor in waste, reducing organics in waste, and combinations thereof. More particularly, the invention relates to isolated  Bacillus  strains, and strains having all of the identifying characteristics of these strains, and combinations thereof, for uses comprising the above-mentioned uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry made under 35 U.S.C. § 371 ofPCT International Application No. PCT/US2018/058948, filed Nov. 2, 2018,which claims under 35 U.S.C. § 119(e) the benefit of and priority toU.S. Patent Application No. 62/687,610, filed Jun. 20, 2018, and U.S.Patent Application No. 62/580,926, filed Nov. 2, 2017, the entiredisclosures of which are incorporated by reference herein.

FIELD OF THE DISCLOSURE

The invention relates to treatment of waste with one or moremicroorganisms for the purposes of, including but not limited to,degrading waste, bioremediation of waste, enhancing waste stabilization,reducing contaminants in waste, reducing odor in waste, reducingorganics in waste, and combinations thereof. More particularly, theinvention relates to isolated Bacillus strains, and strains having allof the identifying characteristics of these strains, and combinationsthereof, for uses comprising the above-mentioned uses.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to microorganisms for use in wastetreatment including, but not limited to, the bioremediation ofindustrial waste, municipal solid waste, landfill waste, soil waste,wastewater, composting waste, contaminated groundwater, leachate fromwaste, polymer-containing waste, hydrocarbon-containing waste, aplastic-containing waste, a polyethylene-containing waste, a highdensity polyethylene-containing waste and plastic bag-containing waste,and methods of their use (e.g., the bioaugmentation of contaminatedwaste, soil, and water). The ability of microbial strains, such asBacillus strains, to produce beneficial enzymes, and their antimicrobialactivity and environmental compatibility, have led to the use of thesemicrobial strains in waste treatment. For example, beneficial microbialstrains can be used to reestablish the balance of bacteria beneficial tothe environment and degrade harmful organic compounds in waste.

As plastics have been in production for less than 200 years, littleinformation has been obtained concerning the rate of decomposition.Polymer crystallinity can limit chain movement and decreases theavailability to degradative agents (including microbial enzymes) andincreases hydrophobicity. Plastic oxidation and degradation can beinitiated by ultraviolet photodegradation, thermooxidation, or bymicrobial or fungal-biosurfactant/enzyme production and biofilmformation. Following oxidation and the breakdown of weakened polymerchains, microorganisms and fungi are able to metabolize the plastic andconvert the carbon to carbon dioxide.

Applicant has developed Bacillus strains, and combinations thereof, thatare useful for waste treatment, waste degradation (includingplastic-containing waste) and controlling the detrimental effects ofwaste, such as by removing a pollutant. These strains can increase therate of decay of plastic waste (e.g., high-density polyethylene),degrade municipal solid waste, enhance waste stabilization, reducecontaminants in waste, reduce chemical oxygen demand, reduce organics inwaste (e.g., hydrocarbons), reduce odor in waste (e.g., hydrogen sulfideand sulfate), and the like. In one embodiment a method of treating wasteto remove a pollutant is provided. The method comprises contacting thewaste with an effective amount of an isolated Bacillus strain selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof, and removing thepollutant.

In another embodiment, a method of controlling a detrimental effect ofwaste is provided. The method comprises contacting the waste with aneffective amount of an isolated Bacillus strain selected from the groupconsisting of Bacillus strain 8992 (NRRL No. B-67472), a strain havingall of the identifying characteristics of Bacillus strain 8992 (NRRL No.B-67472), Bacillus strain 2112 (NRRL No. B-67473), a strain having allof the identifying characteristics of Bacillus strain 2112 (NRRL No.B-67473), Bacillus strain 4954 (NRRL No. B-67474), a strain having allof the identifying characteristics of Bacillus strain 4954 (NRRL No.B-67474), Bacillus strain 2310 (NRRL No. B-67471), a strain having allof the identifying characteristics of Bacillus strain 2310 (NRRL No.B-67471), and combinations thereof, and controlling the detrimentaleffect of the waste.

In various other embodiments, a commercial package, an additive forwaste, and a composition are provided. The commercial package, additivefor waste, and composition comprise an isolated Bacillus strain selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

The following clauses, and combinations thereof, provide variousadditional illustrative aspects of the invention described herein. Thevarious embodiments described in any other section of this patentapplication, including the section titled “DETAILED DESCRIPTION OFILLUSTRATIVE EMBODIMENTS” and the EXAMPLES are applicable to any of thefollowing embodiments of the invention described in the numbered clausesbelow.

1. A method of treating waste to remove a pollutant, the methodcomprising contacting the waste with an effective amount of an isolatedBacillus strain selected from the group consisting of Bacillus strain8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof, and removing the pollutant.

2. The method of clause 1 wherein the waste is selected from the groupconsisting of industrial waste, municipal solid waste, landfill waste,soil waste, wastewater, composting waste, contaminated groundwater,leachate from waste, polymer-containing waste, hydrocarbon-containingwaste, a plastic-containing waste, a polyethylene-containing waste, ahigh density polyethylene-containing waste, and plastic bag-containingwaste.

3. The method of clause 1 or 2 wherein the pollutant is plastic.

4. The method of any one of clauses 1 to 3 wherein the pollutant is apolyethylene.

5. The method of clause 4 wherein the pollutant is a high-densitypolyethylene.

6. The method of any one of clauses 1 to 5 wherein the pollutant is anorganic compound.

7. The method of clause 6 wherein the organic compound is removed bydegradation.

8. The method of any one of clauses 1 to 2 wherein the pollutant is aninorganic compound.

9. The method of any one of clauses 1 to 8 wherein at least one of theBacillus strains has antimicrobial activity.

10. The method of clause 9 wherein the antimicrobial activity is againstbacteria selected from the group consisting of E. coli, Salmonella,Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.

11. The method of any one of clauses 1 to 10 wherein the Bacillus strainproduces an enzyme selected from the group consisting of an a hydrolase,an oxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicelluase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof.

12. The method of clause 11 wherein the enzyme is an esterase.

13. The method of clause 11 wherein the enzyme is a lipase.

14. The method of any one of clauses 1 to 13 further comprising treatingthe waste with another bacterial strain selected from the groupconsisting of another Bacillus strain, a lactic acid bacterial strain,and combinations thereof.

15. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 8992 (NRRL No. B-67472), or a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472).

16. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2112 (NRRL No. B-67473), or a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473).

17. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471).

18. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 4954 (NRRL No. B-67474), or a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474).

19. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 8992 (NRRL No. B-67472).

20. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2112 (NRRL No. B-67473).

21. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2310 (NRRL No. B-67471).

22. The method of any one of clauses 1 to 21 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁶ CFU/gram of the waste.

23. The method of any one of clauses 1 to 21 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁴ CFU/gram of the waste.

24. The method of any one of clauses 1 to 21 wherein the effectiveamount is an amount greater than about 1.0×10² CFU/gram of the waste toabout 1.0×10³ CFU/gram of the waste.

25. The method of any one of clauses 1 to 24 further comprisingcontacting the waste with an enzyme selected from the group consistingof a hydrolase, an oxioreductase, a galactosidase, an arabinoxylanase, aprotease, a lipase, an esterase, an amylase, a hemicellulase, anarabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, andcombinations thereof.

26. A method of controlling a detrimental effect of waste, the methodcomprising contacting the waste with an effective amount of an isolatedBacillus strain selected from the group consisting of Bacillus strain8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof, and controlling the detrimental effect of thewaste.

27. The method of clause 26 wherein the waste is selected from the groupconsisting of industrial waste, municipal solid waste, landfill waste,soil waste, wastewater, composting waste, contaminated groundwater,leachate from waste, polymer-containing waste, hydrocarbon-containingwaste, a plastic-containing waste, a polyethylene-containing waste, ahigh density polyethylene-containing waste, and plastic bag-containingwaste.

28. The method of clause 26 or 27 wherein the detrimental effect iscaused by a plastic.

29. The method of any one of clauses 26 to 28 wherein the detrimentaleffect is caused by a polyethylene.

30. The method of clause 29 wherein the detrimental effect is caused bya high density polyethylene.

31. The method of any one of clauses 26 to 30 wherein the detrimentaleffect is caused by an organic compound.

32. The method of clause 31 wherein the organic compound is removed bydegradation.

33. The method of any one of clauses 26 to 27 wherein the detrimentaleffect is caused by an inorganic compound.

34. The method of any one of clauses 26 to 33 wherein at least one ofthe Bacillus strains has antimicrobial activity.

35. The method of clause 34 wherein the antimicrobial activity isagainst bacteria selected from the group consisting of E. coli,Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.

36. The method of any one of clauses 26 to 35 wherein the Bacillusstrain produces an enzyme selected from the group consisting of ahydrolase, an oxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicellulase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof.

37. The method of clause 36 wherein the enzyme is an esterase.

38. The method of clause 36 wherein the enzyme is a lipase.

39. The method of any one of clauses 26 to 38 further comprisingtreating the waste with another bacterial strain selected from the groupconsisting of another Bacillus strain, a lactic acid bacterial strain,and combinations thereof.

40. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 8992 (NRRL No. B-67472), or a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472).

41. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2112 (NRRL No. B-67473), or a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473).

42. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471).

43. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 4954 (NRRL No. B-67474), or a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474).

44. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 8992 (NRRL No. B-67472).

45. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2112 (NRRL No. B-67473).

46. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2310 (NRRL No. B-67471).

47. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 4954 (NRRL No. B-67474).

48. The method of any one of clauses 26 to 47 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁶ CFU/gram of the waste.

49. The method of any one of clauses 26 to 47 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10³ CFU/gram of the waste.

50. The method of any one of clauses 26 to 49 further comprisingcontacting the waste with an enzyme selected from the group consistingof a hydrolase, an oxioreductase, a galactosidase, an NSPase, a phytase,an arabinoxylanase, a cellulase, a hemicellulase, a protease, anamylase, a xylanase, an esterase, a lipase, and combinations thereof.

51. A commercial package comprising an isolated Bacillus strain selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

52. An additive for waste comprising an isolated Bacillus strainselected from the group consisting of Bacillus strain 8992 (NRRL No.B-67472), a strain having all of the identifying characteristics ofBacillus strain 8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No.B-67473), a strain having all of the identifying characteristics ofBacillus strain 2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No.B-67474), a strain having all of the identifying characteristics ofBacillus strain 4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No.B-67471), a strain having all of the identifying characteristics ofBacillus strain 2310 (NRRL No. B-67471), and combinations thereof.

53. A composition comprising an isolated Bacillus strain selected fromthe group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

54. The commercial package, additive, or composition of any one ofclauses 51 to 53 wherein the Bacillus strain causes degradation of anorganic compound or removal of an inorganic compound in waste.

55. The commercial package, additive, or composition of any one ofclauses 51 54 wherein the Bacillus strain is in the form of aconcentrate.

56. The commercial package, additive, or composition of any one ofclauses 51 to 54 wherein the Bacillus strain is in the form of asuperconcentrate.

57. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the Bacillus strain is in dry form.

58. The commercial package, additive, or composition of any one ofclauses 51 to 57 wherein the Bacillus strain is in pelleted form.

59. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the strain is in a form selected from the groupconsisting of a powder, a liquid, and a pellet form.

60. The commercial package, additive, or composition of any one ofclauses 51 to 59 further comprising a carrier for the Bacillus strain.

61. The commercial package, additive, or composition of clause 60wherein the carrier is selected from the group consisting of salt, adextrin, and combinations thereof.

62. The commercial package, additive, or composition of any one ofclauses 51 to 61 in a bag.

63. The commercial package, additive, or composition of clause 62wherein the bag is a plastic bag.

64. The commercial package, additive, or composition of any one ofclauses 51 to 63 further comprising instructions for use of one or moreof the Bacillus strains.

65. The commercial package, additive, or composition of any one ofclauses 51 to 64 in a 20-pound bag.

66. The commercial package, additive, or composition of any one ofclauses 51 to 64 in a 50-pound bag.

67. The commercial package, additive, or composition of any one ofclauses 51 to 57 or 60 to 66 wherein the Bacillus strain is in powderform.

68. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the Bacillus strain is in liquid form.

69. The commercial package, additive, or composition of any one ofclauses 51 to 68 wherein the Bacillus strain is in a container forcommercial use.

70. The commercial package, additive, or composition of clause 69wherein the container comprises plastic.

71. The commercial package, additive, or composition of clause 69wherein the container comprises paper.

72. The commercial package, additive, or composition of any one ofclauses 51 to 71 further comprising a binder.

73. The commercial package, additive, or composition of clause 72wherein the binder is selected from the group consisting of clay, yeastcell wall components, aluminum silicate, and glucan, or combinationsthereof.

74. The commercial package, additive, or composition of any one ofclauses 51 to 73 wherein the Bacillus strain degrades a plastic.

75. The commercial package, additive, or composition of any one ofclauses 51 to 74 wherein the Bacillus strain degrades a high densitypolyethylene.

76. The method of any one of clauses 26 to 50 wherein the detrimentaleffect is odor and the odor is controlled.

77. The method of clause 76 wherein the odor is caused by sulfate andhydrogen sulfide (H₂S) production.

78. A landfill simulator comprising at least four layers comprising asoil layer, a waste and soil layer, a drainage layer, and a filter.

79. The landfill simulator of clause 78 wherein the soil layer iscompacted soil.

80. The landfill simulator of clause 78 or clause 79 wherein thedrainage layer is granular.

81. The landfill simulator of any one of clauses 78 to 80 wherein thefilter is a geotextile filter.

82. The landfill simulator of any one of clauses 78 to 81 furthercomprising a test sample.

83. The landfill simulator of clause 82 wherein the test sample is abacterium for degrading plastic and wherein the waste in the waste andsoil layer comprises a plastic-containing waste.

84. The landfill simulator of clause 83 wherein the plastic is apolyethylene.

85. The landfill simulator of any one of clauses 78 to 84 wherein thesoil layer is the top layer, the filter is the bottom layer, the wasteand soil layer and the drainage layer are between the soil layer and thefilter, the waste and soil layer is between the soil layer and thedrainage layer, and the drainage layer is between the filter and thewaste and soil layer.

86. The landfill simulator of any one of clauses 82 to 85 for use intesting whether the test sample is capable of degrading plastic.

87. The landfill simulator of any one of clauses 82 to 86 for use intesting how rapidly the test sample is capable of degrading plastic.

88. The landfill simulator of any one of clauses 83 to 87 wherein theplastic is selected from the group consisting of a polyethylene (PE), apolyvinyl chloride (PVC), a polyurethane (PUR), a polystyrene (PS), apolyethylene terephthalate (PET), a polyolefin (PO), an epoxy resin, anelastomer, a thermoplastic, a bio-based plastic, a biodegradableplastic, and a composite plastic.

89. A method of testing whether a test sample can remove a pollutantfrom a landfill or control a detrimental effect of waste in thelandfill, the method comprising the steps of contacting the test samplewith a landfill simulator wherein the landfill simulator comprises atleast four layers comprising a soil layer, a waste and soil layer, adrainage layer, and a filter.

90. The method of clause 89 wherein the soil layer is compacted soil.

91. The method of clause 89 or clause 90 wherein the drainage layer isgranular.

92. The method of any one of clauses 89 to 91 wherein the filter is ageotextile filter.

93. The method of any one of clauses 89 to 92 wherein the test sample isa bacterial strain.

94. The method of clause 93 wherein the method is used to test whetherthe test sample can degrade a plastic and wherein the waste in the wasteand soil layer comprises a plastic-containing waste.

95. The method of clause 94 wherein the plastic is a polyethylene.

96. The method of any one of clauses 89 to 95 wherein the soil layer isthe top layer, the filter is the bottom layer, the waste and soil layerand the drainage layer are between the soil layer and the filter, thewaste and soil layer is between the soil layer and drainage layer, andthe drainage layer is between the filter and the waste and soil layer.

97. The method of any one of clauses 89 to 96 for use in testing howrapidly the test sample is capable of degrading plastic.

98. The method of any one of clauses 94 to 97 wherein the plastic isselected from the group consisting of a polyethylene (PE), a polyvinylchloride (PVC), a polyurethane (PUR), a polystyrene (PS), a polyethyleneterephthalate (PET), a polyolefin (PO), an epoxy resin, an elastomer, athermoplastic, a bio-based plastic, a biodegradable plastic, and acomposite plastic.

99. The method of any one of clauses 89 to 93 for use in testing whetherthe test sample is capable of reducing odor in the landfill.

100. The method of clause 99 wherein the odor is caused by H₂S.

101. The method of any one of clauses 1 to 14 or 22 to 25 wherein thestrain is Bacillus strain 4954 (NRRL No. B-67474).

102. The method of clause 89 wherein the waste is a municipal solidwaste mixture.

103. The method of clause 89 wherein the soil layer comprises syntheticleachate.

104. The method of clause 11 or 36 wherein the enzyme is an amylase.

105. The method of clause 11 or 36 wherein the enzyme is a xylanase.

106. The method of clause 11 or 36 wherein the enzyme is a cellulase.

107. The method of clause 11 or 36 wherein the enzyme is a protease.

108. The method of any one of clauses 1, 2, 9 to 25, 26 to 27, 34 to 50,101, or 104 to 107 wherein the waste is a leachate from a landfill andthe Bacillus strain can be added to the leachate from the landfill as amultiplier to increase dosage rates to the landfill when the leachate isapplied back to the landfill.

109. The method of any one of clauses 1, 2, 3 to 5, 9 to 25, 26 to 30,34 to 50, 101, or 104 to 107 wherein spores of the Bacillus strain areinfused into the plastic.

110. The method of clause 11 or 36 wherein the enzyme is anoxioreductase.

111. The method of clause 11 or 36 wherein the enzyme is agalactosidase.

112. The method of clause 11 or 36 wherein the enzyme is an NSPase.

113. The method of clause 11 or 36 wherein the enzyme is a phytase.

114. The method of clause 11 or 36 wherein the enzyme is anarabinoxylanase.

115. The method of clause 11 or 36 wherein the enzyme is ahemicellulase.

116. The method of clause 11 or 36 wherein the enzyme is a hydrolase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are SEM images comparing the structural physiology of ahigh-density polyethylene (HDPE) strip incubated in a control samplepreparation (1A) and an HDPE strip incubated in Treatment-1 samplepreparation medium (1B), where the physiology of 1B shows dark spots orcavities compared to an absence of dark spots in 1A.

FIGS. 2A and 2B are SEM images comparing the structural physiology of anHDPE strip incubated in a control sample (2A), or an HDPE stripincubated in Treatment-2 sample preparation (2B), and both incubated ina facultative bioreactor landfill simulator, where 2B shows increasedcavitation compared to the control sample in 2A.

FIG. 3 is a schematic of the organization of a landfill simulator.

FIG. 4A-C are SEM images showing rod-shaped particles about 1-2 μm longobserved in all examined areas on the HDPE film consistent with Bacillusmicrobes.

FIG. 5 shows average percent degradation of HDPE samples in minimalmedia for the three sample preparations (n=4, outliers excluded,p=0.0091).

FIGS. 6A and 6B show HDPE average difference (mg) between initial andfinal weight in minimal medium (p=0.047) and average percent degradationof HDPE samples in minimal media for the three sample preparations(p=0.014).

FIGS. 7A and 7B show average difference (mg) between initial and finalHDPE weights in a landfill simulator and average percent degradation ofHDPE in a landfill simulator.

FIG. 8 shows leachate reduction of COD (weeks 2-8) following two monthsincubation in a simulated landfill environment.

FIG. 9 shows leachate reduction of sulfate (weeks 3-8) following twomonths incubation in a simulated landfill environment.

FIG. 10 shows a photograph of a gel displaying a RAPD PCR profile(Primers 2 and 3) for Bacillus strain 8992, Bacillus strain 2112,Bacillus strain 4954, and Bacillus strain 2310.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Applicants have developed Bacillus strains, and combinations thereof,that are useful for waste treatment, waste degradation (includingplastic-containing waste) and controlling the detrimental effects ofwaste, such as by removing a pollutant. These strains can increase therate of decay of plastic waste (e.g., high-density polyethylene),degrade municipal solid waste, enhance waste stabilization, reducecontaminants in waste, reduce chemical oxygen demand, reduce organics inwaste (e.g., hydrocarbons), reduce odor in waste (e.g., hydrogen sulfideand sulfate), and the like. More particularly, the invention relates toisolated Bacillus subtilis and Bacillus amyloliquefaciens strains, andstrains having all of the identifying characteristics of these strains,and combinations thereof, for uses comprising the above-mentioned uses.Bacillus strain 2112 is a Bacillus subtilis strain and strains 8992,4954, and 2310 are Bacillus amyloliquefaciens strains.

In one embodiment a method of treating waste to remove a pollutant isprovided. The method comprises contacting the waste with an effectiveamount of an isolated Bacillus strain selected from the group consistingof Bacillus strain 8992 (NRRL No. B-67472), a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472),Bacillus strain 2112 (NRRL No. B-67473), a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473),Bacillus strain 4954 (NRRL No. B-67474), a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474),Bacillus strain 2310 (NRRL No. B-67471), a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471),and combinations thereof, and removing the pollutant.

In another embodiment, a method of controlling a detrimental effect ofwaste is provided. The method comprises contacting the waste with aneffective amount of an isolated Bacillus strain selected from the groupconsisting of Bacillus strain 8992 (NRRL No. B-67472), a strain havingall of the identifying characteristics of Bacillus strain 8992 (NRRL No.B-67472), Bacillus strain 2112 (NRRL No. B-67473), a strain having allof the identifying characteristics of Bacillus strain 2112 (NRRL No.B-67473), Bacillus strain 4954 (NRRL No. B-67474), a strain having allof the identifying characteristics of Bacillus strain 4954 (NRRL No.B-67474), Bacillus strain 2310 (NRRL No. B-67471), a strain having allof the identifying characteristics of Bacillus strain 2310 (NRRL No.B-67471), and combinations thereof, and controlling the detrimentaleffect of the waste.

In various other embodiments, a commercial package, an additive forwaste, and a composition are provided. The commercial package, additivefor waste, and composition comprise an isolated Bacillus strain selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

The following clauses, and combinations thereof, provide variousadditional illustrative aspects of the invention described herein. Thevarious embodiments described in this section titled “DETAILEDDESCRIPTION OF ILLUSTRATIVE EMBODIMENTS” are applicable to any of thefollowing embodiments of the invention described in the numbered clausesbelow.

1. A method of treating waste to remove a pollutant, the methodcomprising contacting the waste with an effective amount of an isolatedBacillus strain selected from the group consisting of Bacillus strain8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof, and removing the pollutant.

2. The method of clause 1 wherein the waste is selected from the groupconsisting of industrial waste, municipal solid waste, landfill waste,soil waste, wastewater, composting waste, contaminated groundwater,leachate from waste, polymer-containing waste, hydrocarbon-containingwaste, a plastic-containing waste, a polyethylene-containing waste, ahigh density polyethylene-containing waste, and plastic bag-containingwaste.

3. The method of clause 1 or 2 wherein the pollutant is plastic.

4. The method of any one of clauses 1 to 3 wherein the pollutant is apolyethylene.

5. The method of clause 4 wherein the pollutant is a high-densitypolyethylene.

6. The method of any one of clauses 1 to 5 wherein the pollutant is anorganic compound.

7. The method of clause 6 wherein the organic compound is removed bydegradation.

8. The method of any one of clauses 1 to 2 wherein the pollutant is aninorganic compound.

9. The method of any one of clauses 1 to 8 wherein at least one of theBacillus strains has antimicrobial activity.

10. The method of clause 9 wherein the antimicrobial activity is againstbacteria selected from the group consisting of E. coli, Salmonella,Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.

11. The method of any one of clauses 1 to 10 wherein the Bacillus strainproduces an enzyme selected from the group consisting of an a hydrolase,an oxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicelluase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof.

12. The method of clause 11 wherein the enzyme is an esterase.

13. The method of clause 11 wherein the enzyme is a lipase.

14. The method of any one of clauses 1 to 13 further comprising treatingthe waste with another bacterial strain selected from the groupconsisting of another Bacillus strain, a lactic acid bacterial strain,and combinations thereof.

15. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 8992 (NRRL No. B-67472), or a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472).

16. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2112 (NRRL No. B-67473), or a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473).

17. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471).

18. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 4954 (NRRL No. B-67474), or a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474).

19. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 8992 (NRRL No. B-67472).

20. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2112 (NRRL No. B-67473).

21. The method of any one of clauses 1 to 14 wherein the strain isBacillus strain 2310 (NRRL No. B-67471).

22. The method of any one of clauses 1 to 21 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁶ CFU/gram of the waste.

23. The method of any one of clauses 1 to 21 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁴ CFU/gram of the waste.

24. The method of any one of clauses 1 to 21 wherein the effectiveamount is an amount greater than about 1.0×10² CFU/gram of the waste toabout 1.0×10³ CFU/gram of the waste.

25. The method of any one of clauses 1 to 24 further comprisingcontacting the waste with an enzyme selected from the group consistingof a hydrolase, an oxioreductase, a galactosidase, an arabinoxylanase, aprotease, a lipase, an esterase, an amylase, a hemicellulase, anarabinoxylanase, a xylanase, a cellulase, an NSPase, a phytase, andcombinations thereof.

26. A method of controlling a detrimental effect of waste, the methodcomprising contacting the waste with an effective amount of an isolatedBacillus strain selected from the group consisting of Bacillus strain8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof, and controlling the detrimental effect of thewaste.

27. The method of clause 26 wherein the waste is selected from the groupconsisting of industrial waste, municipal solid waste, landfill waste,soil waste, wastewater, composting waste, contaminated groundwater,leachate from waste, polymer-containing waste, hydrocarbon-containingwaste, a plastic-containing waste, a polyethylene-containing waste, ahigh density polyethylene-containing waste, and plastic bag-containingwaste.

28. The method of clause 26 or 27 wherein the detrimental effect iscaused by a plastic.

29. The method of any one of clauses 26 to 28 wherein the detrimentaleffect is caused by a polyethylene.

30. The method of clause 29 wherein the detrimental effect is caused bya high density polyethylene.

31. The method of any one of clauses 26 to 30 wherein the detrimentaleffect is caused by an organic compound.

32. The method of clause 31 wherein the organic compound is removed bydegradation.

33. The method of any one of clauses 26 to 27 wherein the detrimentaleffect is caused by an inorganic compound.

34. The method of any one of clauses 26 to 33 wherein at least one ofthe Bacillus strains has antimicrobial activity.

35. The method of clause 34 wherein the antimicrobial activity isagainst bacteria selected from the group consisting of E. coli,Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.

36. The method of any one of clauses 26 to 35 wherein the Bacillusstrain produces an enzyme selected from the group consisting of ahydrolase, an oxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicellulase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof.

37. The method of clause 36 wherein the enzyme is an esterase.

38. The method of clause 36 wherein the enzyme is a lipase.

39. The method of any one of clauses 26 to 38 further comprisingtreating the waste with another bacterial strain selected from the groupconsisting of another Bacillus strain, a lactic acid bacterial strain,and combinations thereof.

40. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 8992 (NRRL No. B-67472), or a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472).

41. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2112 (NRRL No. B-67473), or a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473).

42. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471).

43. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 4954 (NRRL No. B-67474), or a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474).

44. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 8992 (NRRL No. B-67472).

45. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2112 (NRRL No. B-67473).

46. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 2310 (NRRL No. B-67471).

47. The method of any one of clauses 26 to 39 wherein the strain isBacillus strain 4954 (NRRL No. B-67474).

48. The method of any one of clauses 26 to 47 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10⁶ CFU/gram of the waste.

49. The method of any one of clauses 26 to 47 wherein the effectiveamount of the Bacillus strain is about 1.0×10² CFU/gram of the waste toabout 1.0×10³ CFU/gram of the waste.

50. The method of any one of clauses 26 to 49 further comprisingcontacting the waste with an enzyme selected from the group consistingof a hydrolase, an oxioreductase, a galactosidase, an NSPase, a phytase,an arabinoxylanase, a cellulase, a hemicellulase, a protease, anamylase, a xylanase, an esterase, a lipase, and combinations thereof.

51. A commercial package comprising an isolated Bacillus strain selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

52. An additive for waste comprising an isolated Bacillus strainselected from the group consisting of Bacillus strain 8992 (NRRL No.B-67472), a strain having all of the identifying characteristics ofBacillus strain 8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No.B-67473), a strain having all of the identifying characteristics ofBacillus strain 2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No.B-67474), a strain having all of the identifying characteristics ofBacillus strain 4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No.B-67471), a strain having all of the identifying characteristics ofBacillus strain 2310 (NRRL No. B-67471), and combinations thereof.

53. A composition comprising an isolated Bacillus strain selected fromthe group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof.

54. The commercial package, additive, or composition of any one ofclauses 51 to 53 wherein the Bacillus strain causes degradation of anorganic compound or removal of an inorganic compound in waste.

55. The commercial package, additive, or composition of any one ofclauses 51 54 wherein the Bacillus strain is in the form of aconcentrate.

56. The commercial package, additive, or composition of any one ofclauses 51 to 54 wherein the Bacillus strain is in the form of asuperconcentrate.

57. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the Bacillus strain is in dry form.

58. The commercial package, additive, or composition of any one ofclauses 51 to 57 wherein the Bacillus strain is in pelleted form.

59. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the strain is in a form selected from the groupconsisting of a powder, a liquid, and a pellet form.

60. The commercial package, additive, or composition of any one ofclauses 51 to 59 further comprising a carrier for the Bacillus strain.

61. The commercial package, additive, or composition of clause 60wherein the carrier is selected from the group consisting of salt, adextrin, and combinations thereof.

62. The commercial package, additive, or composition of any one ofclauses 51 to 61 in a bag.

63. The commercial package, additive, or composition of clause 62wherein the bag is a plastic bag.

64. The commercial package, additive, or composition of any one ofclauses 51 to 63 further comprising instructions for use of one or moreof the Bacillus strains.

65. The commercial package, additive, or composition of any one ofclauses 51 to 64 in a 20-pound bag.

66. The commercial package, additive, or composition of any one ofclauses 51 to 64 in a 50-pound bag.

67. The commercial package, additive, or composition of any one ofclauses 51 to 57 or 60 to 66 wherein the Bacillus strain is in powderform.

68. The commercial package, additive, or composition of any one ofclauses 51 to 56 wherein the Bacillus strain is in liquid form.

69. The commercial package, additive, or composition of any one ofclauses 51 to 68 wherein the Bacillus strain is in a container forcommercial use.

70. The commercial package, additive, or composition of clause 69wherein the container comprises plastic.

71. The commercial package, additive, or composition of clause 69wherein the container comprises paper.

72. The commercial package, additive, or composition of any one ofclauses 51 to 71 further comprising a binder.

73. The commercial package, additive, or composition of clause 72wherein the binder is selected from the group consisting of clay, yeastcell wall components, aluminum silicate, and glucan, or combinationsthereof.

74. The commercial package, additive, or composition of any one ofclauses 51 to 73 wherein the Bacillus strain degrades a plastic.

75. The commercial package, additive, or composition of any one ofclauses 51 to 74 wherein the Bacillus strain degrades a high densitypolyethylene.

76. The method of any one of clauses 26 to 50 wherein the detrimentaleffect is odor and the odor is controlled.

77. The method of clause 76 wherein the odor is caused by sulfate andhydrogen sulfide (H₂S) production.

78. A landfill simulator comprising at least four layers comprising asoil layer, a waste and soil layer, a drainage layer, and a filter.

79. The landfill simulator of clause 78 wherein the soil layer iscompacted soil.

80. The landfill simulator of clause 78 or clause 79 wherein thedrainage layer is granular.

81. The landfill simulator of any one of clauses 78 to 80 wherein thefilter is a geotextile filter.

82. The landfill simulator of any one of clauses 78 to 81 furthercomprising a test sample.

83. The landfill simulator of clause 82 wherein the test sample is abacterium for degrading plastic and wherein the waste in the waste andsoil layer comprises a plastic-containing waste.

84. The landfill simulator of clause 83 wherein the plastic is apolyethylene.

85. The landfill simulator of any one of clauses 78 to 84 wherein thesoil layer is the top layer, the filter is the bottom layer, the wasteand soil layer and the drainage layer are between the soil layer and thefilter, the waste and soil layer is between the soil layer and thedrainage layer, and the drainage layer is between the filter and thewaste and soil layer.

86. The landfill simulator of any one of clauses 82 to 85 for use intesting whether the test sample is capable of degrading plastic.

87. The landfill simulator of any one of clauses 82 to 86 for use intesting how rapidly the test sample is capable of degrading plastic.

88. The landfill simulator of any one of clauses 83 to 87 wherein theplastic is selected from the group consisting of a polyethylene (PE), apolyvinyl chloride (PVC), a polyurethane (PUR), a polystyrene (PS), apolyethylene terephthalate (PET), a polyolefin (PO), an epoxy resin, anelastomer, a thermoplastic, a bio-based plastic, a biodegradableplastic, and a composite plastic.

89. A method of testing whether a test sample can remove a pollutantfrom a landfill or control a detrimental effect of waste in thelandfill, the method comprising the steps of contacting the test samplewith a landfill simulator wherein the landfill simulator comprises atleast four layers comprising a soil layer, a waste and soil layer, adrainage layer, and a filter.

90. The method of clause 89 wherein the soil layer is compacted soil.

91. The method of clause 89 or clause 90 wherein the drainage layer isgranular.

92. The method of any one of clauses 89 to 91 wherein the filter is ageotextile filter.

93. The method of any one of clauses 89 to 92 wherein the test sample isa bacterial strain.

94. The method of clause 93 wherein the method is used to test whetherthe test sample can degrade a plastic and wherein the waste in the wasteand soil layer comprises a plastic-containing waste.

95. The method of clause 94 wherein the plastic is a polyethylene.

96. The method of any one of clauses 89 to 95 wherein the soil layer isthe top layer, the filter is the bottom layer, the waste and soil layerand the drainage layer are between the soil layer and the filter, thewaste and soil layer is between the soil layer and drainage layer, andthe drainage layer is between the filter and the waste and soil layer.

97. The method of any one of clauses 89 to 96 for use in testing howrapidly the test sample is capable of degrading plastic.

98. The method of any one of clauses 94 to 97 wherein the plastic isselected from the group consisting of a polyethylene (PE), a polyvinylchloride (PVC), a polyurethane (PUR), a polystyrene (PS), a polyethyleneterephthalate (PET), a polyolefin (PO), an epoxy resin, an elastomer, athermoplastic, a bio-based plastic, a biodegradable plastic, and acomposite plastic.

99. The method of any one of clauses 89 to 93 for use in testing whetherthe test sample is capable of reducing odor in the landfill.

100. The method of clause 99 wherein the odor is caused by H₂S.

101. The method of any one of clauses 1 to 14 or 22 to 25 wherein thestrain is Bacillus strain 4954 (NRRL No. B-67474).

102. The method of clause 89 wherein the waste is a municipal solidwaste mixture.

103. The method of clause 89 wherein the soil layer comprises syntheticleachate.

104. The method of clause 11 or 36 wherein the enzyme is an amylase.

105. The method of clause 11 or 36 wherein the enzyme is a xylanase.

106. The method of clause 11 or 36 wherein the enzyme is a cellulase.

107. The method of clause 11 or 36 wherein the enzyme is a protease.

108. The method of any one of clauses 1, 2, 9 to 25, 26 to 27, 34 to 50,101, or 104 to 107 wherein the waste is a leachate from a landfill andthe Bacillus strain can be added to the leachate from the landfill as amultiplier to increase dosage rates to the landfill when the leachate isapplied back to the landfill.

109. The method of any one of clauses 1, 2, 3 to 5, 9 to 25, 26 to 30,34 to 50, 101, or 104 to 107 wherein spores of the Bacillus strain areinfused into the plastic.

110. The method of clause 11 or 36 wherein the enzyme is anoxioreductase.

111. The method of clause 11 or 36 wherein the enzyme is agalactosidase.

112. The method of clause 11 or 36 wherein the enzyme is an NSPase.

113. The method of clause 11 or 36 wherein the enzyme is a phytase.

114. The method of clause 11 or 36 wherein the enzyme is anarabinoxylanase.

115. The method of clause 11 or 36 wherein the enzyme is ahemicellulase.

116. The method of clause 11 or 36 wherein the enzyme is a hydrolase.

In various embodiments, the Bacillus strain (e.g., Bacillus strain 8992(NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), or Bacillus strain 2310 (NRRL No.B-67471) for use in accordance with the methods, commercial packages,additives for waste, and compositions described herein can be selectedfrom the group consisting of Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof. Bacillus strains 89922112, 4954, and 2310 were deposited on Jun. 27, 2017 at the AgriculturalResearch Service Culture Collection (NRRL), International DepositoryAuthority, 1815 North University Street, Peoria, Ill. 61604-3999, andwere given accession numbers NRRL No. B-67472, NRRL No. B-67473, NRRLNo. B-67474, and NRRL No. B-67471, respectively. The deposits were madeunder the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. The NRRL strain designations are MDG-8992, MDG-2112,MDG-4954, and MDG-2310, which are equivalent to Bacillus strain 8992,2112, 4954, and 2310, respectively, as referred to in the application.

Any of these strains can be used to treat waste alone or in combinationin the form of an additive for waste or a composition as describedherein (e.g., an additive or a composition further comprising a carrierand/or a binder). In one embodiment, multiple strains are used to treatwaste in combination in a single composition. In another embodiment,multiple strains are used to treat waste in combination in separatecompositions.

As used herein “a strain having all of the identifying characteristicsof” Bacillus strain 8992, 2112, 4954, or 2310 can be a mutant strainhaving all of the identifying characteristics of Bacillus strain 8992,2112, 4954, or 2310 (e.g., a DNA fingerprint based on DNA analysis thatcorresponds to the DNA fingerprint of Bacillus strain 8992, 2112, 4954,or 2310, enzyme activities that correspond to Bacillus strain 8992,2112, 4954, or 2310, antimicrobial activity that corresponds to Bacillusstrain 8992, 2112, 4954, or 2310, antibiotic sensitivity and toleranceprofiles that correspond to Bacillus strain 8992, 2112, 4954, or 2310,or combinations thereof). In alternate embodiments, the mutation can bea natural mutation, or a genetically engineered mutation. In anotherembodiment, “a strain having all of” the identifying characteristics ofBacillus strain 8992, 2112, 4954, or 2310 can be a strain, for example,produced by isolating one or more plasmids from Bacillus strain 8992,2112, 4954, or 2310 and introducing the one or more plasmids intoanother bacterium, such as another Bacillus strain, as long as the oneor more plasmids contain DNA that provides the identifyingcharacteristics of Bacillus strain 8992, 2112, 4954, or 2310 (e.g., aDNA fingerprint based on DNA analysis that corresponds to the DNAfingerprint of Bacillus strain 8992, 2112, 4954, or 2310).

In another embodiment, one or more of the Bacillus strains described inthe preceding paragraphs (e.g., Bacillus strain 8992, 2112, 4954, or2310) can be used to treat waste along with another bacterial strainselected from the group consisting of another Bacillus strain, a lacticacid bacterial strain, and combinations thereof. In still anotherembodiment, the additional Bacillus strain can be selected from thegroup consisting of Bacillus subtilis, Bacillus amyloliquefaciens,Bacillus licheniformis, Bacillus pumilus, Bacillus mojavensis, Bacillusmethylotrophicus, other Bacillus strains, and combinations thereof. Inyet another embodiment, one or more of the Bacillus strains described inthe preceding paragraphs (e.g., Bacillus strain 8992, 2112, 4954, or2310) can be used to treat waste along with any other bacterial straineffective to treat waste to remove pollutants or to control detrimentaleffects of waste

The additive for waste or the composition described herein can be usedto treat waste for any period of time that is effective to removepollutants and/or control the detrimental effects of waste. For example,in one embodiment treatment of waste can occur daily. The time periodsfor treatment of waste are non-limiting and it should be appreciatedthat any time period or treatment schedule determined to be effective toremove pollutants and/or control the detrimental effects of waste may beused.

In various illustrative embodiments, the Bacillus strain (e.g., Bacillusstrain 8992, 2112, 4954, and/or 2310), or any other bacterial strainsadded in addition to Bacillus strain 8992, 2112, 4954, and/or 2310, canbe added to the waste at about 1.0×10² CFU/gram of the waste, about1.0×10² CFU/gram of the waste to about 1.0×10³ CFU/gram of the waste,about 1.0×10² CFU/gram of the waste to about 1.0×10⁴ CFU/gram of thewaste, about 1.0×10² CFU/gram of the waste to about 1.0×10⁵ CFU/gram ofthe waste, about 1.0×10² CFU/gram of the waste to about 1.0×10⁶ CFU/gramof the waste, about 1.0×10³ CFU/gram of the waste to about 5.0×10¹²CFU/gram of the waste or at about 1.0×10³ CFU/gram of the waste to about1.0×10¹⁰ CFU/gram of the waste. In other embodiments, the Bacillusstrain (e.g., Bacillus strain 8992, 2112, 4954, and/or 2310) can beadded to the waste at an amount greater than about 1.0×10² CFU/gram ofthe waste, at greater than about 1.0×10³ CFU/gram of the waste, atgreater than about 1.1×10³ CFU/gram of the waste, at greater than about1.25×10³ CFU/gram of the waste, at greater than about 1.5×10³ CFU/poundof the waste, at greater than about 1.75×10³ CFU/gram of the waste, atgreater than about 1.0×10⁴ CFU/gram of the waste, at greater than about2.0×10⁴ CFU/gram of the waste, at greater than about 3.0×10⁴ CFU/gram ofthe waste, at greater than about 4.0×10⁴ CFU/gram of the waste, atgreater than about 5.0×10⁴ CFU/gram of the waste, at greater than about6.0×10⁴ CFU/gram of the waste, at greater than about 7.0×10⁴ CFU/gram ofthe waste, at greater than about 8.0×10⁴ CFU/gram of the waste, atgreater than about 1.0×10⁵ CFU/gram of the waste, at greater than about1.0×10⁶ CFU/gram of the waste, at greater than about 1.0×10⁷ CFU/gram ofthe waste, at greater than about 1.0×10⁸ CFU/gram of the waste, atgreater than about 1.0×10⁹ CFU/gram of the waste, at greater than about1.0×10¹⁰ CFU/gram of the waste, at greater than about 1.0×10¹¹ CFU/gramof the waste, or at greater than about 1.0×10¹² CFU/gram of the waste.

In various embodiments, the waste described herein can be selected fromthe group consisting of industrial waste, municipal solid waste,landfill waste, soil waste, wastewater, composting waste, contaminatedgroundwater, leachate from waste, polymer-containing waste,hydrocarbon-containing waste, a plastic-containing waste, apolyethylene-containing waste, a high density polyethylene-containingwaste, and plastic bag-containing waste. In another embodiment, theplastic-containing waste can be a polyethylene-containing waste (e.g., ahigh-density polyethylene), or any other type of waste comprisingpollutants that need to be removed or having a detrimental effect thatneeds to be controlled. In another embodiment, the plastic-containingwaste can be selected from the group consisting of a polyethylene (PE),a polyvinyl chloride (PVC), a polyurethane (PUR), a polystyrene (PS), apolyethylene terephthalate (PET), a polyolefin (PO), an epoxy resin, anelastomer, a thermoplastic, a bio-based plastic, a biodegradableplastic, and a composite plastic.

In another embodiment where the waste is leachate from a landfill, theBacillus strain can be added to the leachate from a landfill as amultiplier to increase dosage rates to the landfill when the leachate isapplied back to the landfill (i.e., the strains multiply in the leachateso the dose that is applied back to the landfill is increased). In yetanother embodiment spores from the Bacillus strain can be infused intoplastic (e.g., plastic bag-containing waste and plastic-containingwaste) to treat the plastic with the Bacillus strain.

As used herein “remove a pollutant” or “removal of a pollutant” meanscompletely removing the pollutant, reducing the amount of the pollutant,inactivating the pollutant, degrading the pollutant, or causing thepollutant to be converted to an inactivated form. As used herein“controlling a detrimental effect of waste” or similar phrases meanscompletely removing a pollutant causing the detrimental effect, reducingthe amount of the pollutant causing the detrimental effect, inactivatingthe pollutant causing the detrimental effect, degrading the pollutantcausing the detrimental effect, or causing the pollutant responsible forthe detrimental effect to be converted to an inactivated form.“Controlling a detrimental effect of waste” can mean degrading waste,enhancing waste stabilization, reducing contaminants in waste, reducingCOD, reducing organics in waste (e.g., hydrocarbons), reducing odor inwaste (e.g., H₂S and sulfate), and the like.

In various illustrative aspects, the pollutants that can be removed fromthe waste can be selected from the group consisting of a harmfulmicroorganism, an organic compound, an inorganic compound, aplastic-containing compound, a polyethelene-containing compound, ahigh-density polyethelene-containing compound, and combinations thereof,and the like. In some embodiments described herein, at least one of theBacillus strains can have antimicrobial activity. Such antimicrobialactivity can be against, for example, E. coli, Salmonella,Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.

In the embodiment where the pollutant is an organic compound, theorganic compound can be removed by degradation. In this embodiment, theorganic compound can be, for example, a plastic-containing compound, apolyethelene-containing compound, a high-density polyethelene-containingcompound, and combinations thereof, and the like, or a by-product of anyother organic compound that is a pollutant in waste or is the cause of adetrimental effect of waste. In the embodiment where the pollutant is anorganic compound, the organic compound can be from, for example, agrocery bag, or any other waste that is plastic-containing waste,including a polyethelene-containing waste or a high-densitypolyethelene-containing waste.

In various illustrative aspects, the Bacillus strains described herein(i.e., Bacillus strains 8992, 2112, 4954, and/or 2310) produce an enzymeselected from the group consisting of a hydrolase, an oxioreductase, agalactosidase, an NSPase, a phytase, an arabinoxylanase, a cellulase, ahemicellulase, a protease, an amylase, a xylanase, an esterase, alipase, and combinations thereof.

In one illustrative embodiment, one or more enzymes may be added to theadditive for waste or the composition described herein or may be addeddirectly to the waste in combination with the Bacillus strains describedherein. In various embodiments, the enzymes that may be used to treatthe waste in addition to the Bacillus strains include a hydrolase, anoxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicellulase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof, and any otherenzyme that is suitable to treat waste to remove pollutants or control adetrimental effect of waste. Any of the enzymes described above that aresuitable for treatment of waste may be added as a component of thecommercial package, additive for waste, or composition described herein,or may be added directly the waste as a separate composition.

In additional embodiments of the invention, compositions comprisingBacillus strain 8992, 2112, 4954, and/or 2310 are provided. In oneembodiment, a commercial package is provided comprising an isolatedBacillus strain selected from the group consisting of Bacillus strain8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof.

In another embodiment, an additive for waste is provided comprising anisolated Bacillus strain selected from the group consisting of Bacillusstrain 8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof.

In yet another embodiment, a composition is provided comprising anisolated Bacillus strain selected from the group consisting of Bacillusstrain 8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), a strain having all of the identifyingcharacteristics of Bacillus strain 2310 (NRRL No. B-67471), andcombinations thereof.

In these embodiments the Bacillus strain can be in the form of, forexample, a powder, a liquid, or pellets, and can be mixed with the wasteusing any suitable method known in the art to achieve any of the amountsof Bacillus strain 8992 (NRRL No. B-67472), a strain having all of theidentifying characteristics of Bacillus strain 8992 (NRRL No. B-67472),Bacillus strain 2112 (NRRL No. B-67473), a strain having all of theidentifying characteristics of Bacillus strain 2112 (NRRL No. B-67473),Bacillus strain 4954 (NRRL No. B-67474), a strain having all of theidentifying characteristics of Bacillus strain 4954 (NRRL No. B-67474),Bacillus strain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471),and combinations thereof, for the treatment of waste to removepollutants or to control a detrimental effect of waste.

In any of the composition embodiments described herein, the Bacillusstrain 8992 (NRRL No. B-67472), a strain having all of the identifyingcharacteristics of Bacillus strain 8992 (NRRL No. B-67472), Bacillusstrain 2112 (NRRL No. B-67473), a strain having all of the identifyingcharacteristics of Bacillus strain 2112 (NRRL No. B-67473), Bacillusstrain 4954 (NRRL No. B-67474), a strain having all of the identifyingcharacteristics of Bacillus strain 4954 (NRRL No. B-67474), Bacillusstrain 2310 (NRRL No. B-67471), or a strain having all of theidentifying characteristics of Bacillus strain 2310 (NRRL No. B-67471),and combinations thereof, can inhibit a pathogen selected from the groupconsisting of E. coli, Salmonella, Staphylococcus, Enterococcus,Clostridia, Campylobacter, and combinations thereof. These types ofmicroorganisms are non-limiting examples of the types of microorganismsthe Bacillus strains can inhibit.

In these embodiments, the Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), or astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof, can cause degradationor removal of an organic compound or removal of an inorganic compound inwaste.

In illustrative aspects, the Bacillus strain 8992 (NRRL No. B-67472), astrain having all of the identifying characteristics of Bacillus strain8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), astrain having all of the identifying characteristics of Bacillus strain2112 (NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), astrain having all of the identifying characteristics of Bacillus strain4954 (NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), or astrain having all of the identifying characteristics of Bacillus strain2310 (NRRL No. B-67471), and combinations thereof, can be in the form ofa commercial package, an additive for waste or any suitable composition.In another illustrative embodiment, the Bacillus strain(s) in thecommercial package, additive for waste, or composition can be in theform of a concentrate (e.g., about 1×10⁸ to about 5×10⁹ CFU/g) or asuperconcentrate (e.g., about 1×10¹⁰ to about 5×10¹² CFU/g). In anotherembodiment, the Bacillus strain(s) in the commercial package, additivefor waste, or composition can be in a dry form (e.g., a powder), apelleted form, a liquid form, a freeze-dried form, or in the form of agel, or any other suitable form.

In another illustrative embodiment, the Bacillus strain(s) in thecommercial package, additive for waste, or composition can furthercomprise a carrier for the Bacillus strain(s). In various embodiments,the carrier can be selected from the group consisting of a salt, mineraloil, a dextrin (e.g., maltodextrin), whey, sugar, limestone, driedstarch, sodium silico aluminate, and combinations thereof. In anotherembodiment, the carrier can be any suitable carrier known in the art fora composition for treating waste. In another embodiment, the Bacillusstrain(s) in the commercial package, additive for waste, or compositioncan further comprise a binder such as clay, yeast cell wall components,aluminum silicate, glucan, or other known binders, and/ormicronutrients, including but not limited to, nitrogen and phosphorus.

In yet other embodiments, the commercial package, additive for waste, orcomposition comprising Bacillus strain 8992 (NRRL No. B-67472), a strainhaving all of the identifying characteristics of Bacillus strain 8992(NRRL No. B-67472), Bacillus strain 2112 (NRRL No. B-67473), a strainhaving all of the identifying characteristics of Bacillus strain 2112(NRRL No. B-67473), Bacillus strain 4954 (NRRL No. B-67474), a strainhaving all of the identifying characteristics of Bacillus strain 4954(NRRL No. B-67474), Bacillus strain 2310 (NRRL No. B-67471), or a strainhaving all of the identifying characteristics of Bacillus strain 2310(NRRL No. B-67471), and combinations thereof, is in a container forcommercial use. In various embodiments the container can be, forexample, a bag (e.g., a 20-pound bag, a 50-pound bag, a 2-ounce bag, a1-pound bag, or a 1-kilogram bag), a pouch, a drum, a bottle, or a box.In illustrative aspects, the container comprising the Bacillus strain(s)can comprise plastic, metal, foil, paper, fiber, or cardboard (e.g., aplastic pail, a paper bag, a foil bag, a fiber drum, etc.). Thecommercial package can further comprise instructions for use of one ormore of the Bacillus strains.

In other illustrative embodiments, a landfill simulator of clauses orclaims 78 to 88 or the method of using a landfill simulator of clausesor claims 89 to 100 can be used to test whether a test sample can removea pollutant from a landfill or control a detrimental effect of waste inthe landfill, the method comprising the steps of contacting the testsample with a landfill simulator wherein the landfill simulatorcomprises at least four layers comprising a soil layer, a waste and soillayer, a drainage layer, and a filter. A diagram of an exemplarylandfill simulator is shown in FIG. 3 .

The following examples are for illustrative purposes only. The examplesare non-limiting, and are not intended to limit the invention in anyway.

Example 1 HDPE in Minimal Media Preparation

Lab-scale testing was conducted twice in minimal media containingpre-weighed high-density polyethylene (HDPE) as a sole carbon source.Three preparations were made in quadruplicate—a control containing onlythe minimal media, micronutrients, and HDPE; Treatment-1 (analogous toCOMBO-1 in the priority document) including the minimal media, MDG-8992,MDG-2112, micronutrients and HDPE; and Treatment-2 (analogous to COMBO-2in the priority document) including the minimal media, MDG-4954,MDG-2310, micronutrients and HDPE. Inoculation and media replacementoccurred weekly over two months (pH=7.0 at 30° C.), increasing the rateof decay of HDPE.

Post incubation, HDPE strips were soaked in a 2% sodium dodecyl sulfate(SDS) solution and washed with deionized water to remove biofilm andcontaminants. The HDPE film was dried at 55° C. for 72 hours and weighedusing a Mettler Toledo Analytical Balance (model number XS104; obtainedfrom Mettler Toledo). Results for the initial minimal medium experimentwere calculated excluding and including outliers (n=4). The microbialdegradation of HDPE was analyzed by measuring the dry-weight reductionof HDPE film showing statistical significance of treatment using one-wayanalysis of variance (ANOVA) and scanning electron microscopy (SEM).Results from combined initial and duplicate experiments includingoutliers (n=8) were also assessed by measuring the dry-weight reductionof HDPE film and one-way ANOVA to determine significance.

Example 2 Results and Analysis from HDPE Experiment in Minimal MediaPreparation

The following calculation was used to calculate the dry-weight reductionof HDPE samples: % mass loss=[(initial weight minus finalweight)/initial weight]×100. Results were analyzed with and withoutoutliers. Outliers were determined using the Iglewicz and Hoaglinoutlier test (1993), “Volume 16: How to Detect and Handle Outliers”, TheASQC Basic References in Quality Control: Statistical Techniques, EdwardF. Mykytka, Ph.D., Editor. Results for the initial minimal mediaexperiment excluding outliers (n=4) show degradation at 0.09%±0.05,0.52%±0.19 and 0.43%±0.04 for control, treatment-1, and treatment-2respectively (FIG. 5 ). Assuming a simple linear rate of degradation,the projected time for HDPE materials to degrade completely was 171years, 30 years and 36 years for Control, Treatment-1 and Treatment-2preparations, respectively (Table 1).

TABLE 1 Projected time to complete degradation given constant rate.Sample % Average Years to Degrade Control 0.09 171 Treatment-1 0.52 30Treatment-2 0.43 36

Statistical significance of treatment on the difference between initialand final HDPE weights was calculated using one-way ANOVA. When thep-value is lower than 0.05, one or more treatments are significantlydifferent. One-way ANOVA for percent degradation of HDPE excludingoutliers had a p-value at p=0.0091 and f-statistic at f=9.9180. One-wayANOVA for the milligram difference between initial and final weightsincluding outliers had p=0.004. One-way ANOVA fit to a general linearmodel (GLM) for the milligram difference between initial and finalweights using initial weight as a covariate had p=0.006.

Scanning Electron Microscopy images were taken of an un-treated HDPEreference sample (FIG. 1A) and test sample (FIG. 1B). The test samplewas incubated for two-months in minimal media (pH=7.0 at 30° C.) withweekly inoculation and media replacement identical to the Treatment-2preparation. SEM imaging showed both reference and test samples ashaving similarly rough surfaces with many small bright particles and asomewhat fibrous morphology. SEM images of the reference sample showedno visible pocketing or cavitation. SEM images of the test sample showedvisible changes to the surface morphology of the HDPE material (whencompared to the reference sample) with increased cavitation, potentiallydue to microbes consuming or burrowing into the HDPE film.

Results from combined initial and duplicate experiments (n=8) includingoutliers were assessed using one-way analysis of variance (ANOVA) todetermine the statistical significance of treatment on the differencebetween initial and final HDPE weight in milligrams. FIG. 6A shows thedifferences between initial and final HDPE weight show degradation at0.21 mg±0.11, 0.60 mg±0.43 and 0.56 mg±0.35 for control, treatment-1,and treatment-2 respectively (p=0.047). The p-value was stronger whenusing ANOVA fit to a general linear model to compare final weight tosample treatment with initial weight as a covariate. FIG. 6B showsdegradation at 0.19%±0.09, 0.41%±0.17 and 0.41%±0.15 for control,treatment-1, and treatment-2 respectively (p=0.014; control vs.treatment-1 p=0.016; control vs. treatment-2 p=0.008).

Example 3 HDPE Decay in Landfill Simulator Model Preparation

A pilot-scale landfill simulator was constructed to study degradativeeffect of microbial and nutritional amendments on HDPE. The simulatorwas constructed using six cylinders loaded with a sandy brown loam soiland a granular layer separated by landscaping fabric (FIG. 3 ). Evolvedleachate was collected in reservoirs and analyzed weekly using Hach TNTplus chemistries for changes in chemical oxygen demand (COD) andsulfate. Following chemical analysis, leachate was amended andrecirculated for all samples.

Three preparations were made in duplicate—a Control containing only theminimal media, micronutrients and HDPE; Treatment-1 including theminimal media, MDG-8992, MDG-2112, micronutrients and HDPE; andTreatment-2 including the minimal media, MDG-4954, MDG-2310,micronutrients and HDPE. Pre-weighed HDPE film samples were surroundedby a proprietary blend of municipal solid waste, which was thensuspended and compressed in the lower third of the soil-filledcylinders. Moisture levels were adjusted to field capacity and aproprietary blend of synthetic leachate was added to initial moistureamendments. Cylinders were incubated for two months at room temperaturebetween 18 and 34° C. (pH=7.35±0.3). Evolved leachate (200 mL) wasrecirculated daily and amended weekly. Temperature and pH were recordedprior to and following leachate amendments. Amendments included water(adjusted to 200 mL), a pH buffer, a proprietary blend of micronutrientsand the inoculation of treated preparations.

To remove biofilm and clean, HDPE film samples were soaked in a 2%sodium dodecyl sulfate (SDS) solution, washed with tap water, and rinsedwith both 70% ethanol and deionized water. The film was dried at 55° C.for 72 hours and weighed on an analytical balance. The microbialdegradation of HDPE was analyzed by measuring the dry-weight reductionof HDPE film, imaging with a scanning electron microscopy (SEM) andsurface profile characterization using scanning white light interferencemicroscopy (SWLIM).

Example 4 Results and Analysis from Landfill Simulator Model Preparation

The following calculation was used to calculate the dry-weight reductionof HDPE samples: % mass loss=[(initial weight minus finalweight)/initial weight]×100. FIG. 7A shows the average differencesbetween initial and final weight (mg) from samples incubated in thelandfill simulator (n=2) show a decrease in weight at 1.5 mg, 2.15 mgand 1.70 mg. Percent degradation of HDPE film was 0.75%, 1.07% and 1.06%for control, treatment-1 and Treatment-2 respectively (FIG. 7B). Thedegradation rate in treated samples increased 30%.

Scanning Electron Microscopy images of HDPE film from the landfillsimulator experiment were taken of Control and Treatment-2 samples (onereplicate of the two) following incubation in the landfill simulator.Control sample (FIG. 2A) showed decreased cavitation when compared tothe test sample (FIG. 2B), potentially due to microbes consuming orburrowing into the HDPE film. HDPE in the Control showed some visiblepocketing and cavitation due to the non-sterile environment. Resultsfrom the surface profile characterization using SWLIM showed increasedroughness, higher peaks and lower average valley depth in the testsample when compared to the control (Table 2).

TABLE 2 Results from surface profile characterization using scanningwhite light interference microscopy (SWLIM) show increased roughness,higher peaks and lower average valley depth in the test sample whencompared to the control. MVA12005 Sample Ra *Rku Rp Rpm Rq *Rsk Rt RvRvm Rz Test Sample 12005ac0439_1 330.8 3.1 1844.8 1654.5 416.4 0.24673.5 −2828.6 −2069.0 3723.5 12005ac0439_2 317.6 3.7 2072.5 1891.4407.2 0.3 4792.0 −2719.5 −2051.3 3942.7 12005ac0439_3 328.2 3.1 1621.91515.8 412.3 0.2 4204.9 −2583.0 −2020.1 3535.7 12005ac0439_4 357.6 3.32102.6 1878.5 453.5 0.2 5161.2 −3058.6 −2356.2 4234.7 12005ac0439_5330.1 3.3 2080.5 1881.6 416.2 0.3 5271.6 −3191.1 −1899.0 3780.612005ac0439_6 418.2 4.6 3812.8 3095.9 542.1 0.4 8137.9 −4325.1 −2735.25831.1 12005ac0439_7 650.3 2.9 2888.3 2676.7 810.8 0.0 6448.4 −3560.1−2644.4 5321.1 12005ac0439_8 335.8 8.8 3610.8 3256.8 455.4 1.2 5756.6−2146.1 −1749.3 5006.1 12005ac0439_9 367.1 4.2 2916.5 2395.5 472.4 0.55549.5 −2632.9 2048.0 4443.5 12005ac0439_10 548.6 4.0 3865.9 3453.3706.1 0.7 7884.9 −4018.9 −2430.9 5884.2 Average 398.6 4.1 2681.7 2370.0509.2 0.4 5788.1 −3106.4 −2200.3 4570.3 Std. Dev. 112.5 1.7 852.1 709.7139.5 0.4 1325.5 681.5 324.9 882.1 Control Sample 12005ac0440_1 331.33.1 1843.5 1644.0 416.2 0.1 4866.7 −3123.2 −2164.1 3808.1 12005ac0440_2315.0 3.9 2172.6 2047.9 400.4 0.3 6247.8 −4075.2 −2746.8 4794.712005ac0440_3 375.3 3.1 2255.3 1969.0 472.2 0.1 5091.0 −2835.7 −2206.04174.9 12005ac0440_4 442.6 2.8 2306.2 2072.1 550.1 0.3 5063.2 −2757.0−2189.7 4261.9 12005ac0440_5 310.1 3.0 1766.1 1520.0 389.5 0.2 3081.3−2035.2 −1636.6 3156.5 12005ac0440_6 368.1 6.1 3421.0 3138.7 482.7 0.75859.5 −2438.5 −1841.0 4979.7 12005ac0440_7 340.5 3.2 1744.6 1672.8428.4 0.2 5425.5 −3680.9 −2506.8 4179.6 12005ac0440_8 393.6 3.3 2741.02285.0 495.6 0.2 4877.4 −2136.5 −1897.0 4182.0 12005ac0440_9 311.2 3.11966.5 1702.5 389.5 0.2 4651.1 2685.6 −2083.1 3785.6 12005ac0440_10338.5 3.4 2055.8 1978.2 430.5 0.2 4409.6 −2353.7 −1867.0 3743.1 Average352.6 3.5 2227.2 1992.8 445.5 0.2 5039.3 −2812.1 −213.8 4106.6 Std. Dev.42.5 0.9 515.3 465.9 52.9 0.2 699.0 656.3 330.5 527.3 *Rku, Rsk: nounits All other measurements: nm Ra - roughness average Rku - kurtosisRp - maximum profile peak height Rpm - average maximum profile peakheight Rq - root mean square roughness (rms) Rsk - skewness Rt - maximumheight of the profile Rv - maximum profile valley depth Rvm - averagemaximum profile valley depth Rz - average maximum height of the profile

Table 3 includes the projected time to complete degradation assuming asimple linear rate in a simulated landfill environment for each of thesample preparations. The data showed that in a simulated landfillenvironment, complete degradation of HDPE would take 21 years, 14 years,and 15 years for the Control, Treatment-1 and Treatment-2, respectively.When comparing the averaged treated results to the control for landfillsimulator, there is a six-year difference degradation rates for treatedsamples. If pilot testing results could be scaled to field testing, thebioaugmentation of incoming waste could reduce plastic by 30%.

TABLE 3 Projected Time to Complete Degradation Given Constant Rate.Sample % Average Years to Degrade Control 0.75 21 Treatment-1 1.07 14Treatment-2 1.06 15

Table 4 presents a comparison for the average HDPE degradation in yearsif a simple linear rate of degradation is assumed for samples incubatedin minimal media (including the initial experiment excluding outliersand combined experiments including outliers) and a simulated landfillenvironment for all sample preparations (Control, Treatment-1 andTreatment-2). Results from incubation in the landfill simulator show ahigher rate of degradation when compared to incubation in minimal mediadespite decreased moisture content (approximately 12% at fieldcapacity), oxygen, dosage, nutrients and temperature, which could beattributed to the native microbial communities and extra nutrients frommunicipal solid waste in the simulator.

TABLE 4 Results comparing years to degrade assuming a simple linear ratefor the initial experiment in minimal media excluding outliers, combinedduplicate experiments in minimal media including outliers, and resultsfrom incubation in a simulated landfill environment. Initial minimalCombined minimal media Experiment media experiments Landfill simulator(n = 4) (n = 8) (n = 2) Sample % Average Years % Average Years % AverageYears Control 0.09 ± 0.05 171 0.19 ± 0.09 81 0.75 21 Treatment-1 0.52 ±0.19 30 0.41 ± 0.17 38 1.07 14 Treatment-2 0.43 ± 0.04 36 0.41 ± 0.15 381.06 15

Biological and nutritional leachate amendments lowered the COD for allsamples with increased reduction in treated preparations compared to thecontrol (n=2). COD was reduced by 27%, 38% and 41% for control,treatment-1, and treatment-2 respectively (reduction from weeks 2-8 inFIG. 8 ). Sulfate was reduced below detectable levels for all samples ata minimum of 83%, 77%, and 81% for control, treatment-1, and treatment-2respectively (reduction from weeks 3-8 in FIG. 9 ).

In addition to the reduction of plastics, the biological treatment ofleachate, nutrient amendments and recirculation have potential improveleachate quality. When COD and leachate toxicity is decreased, there isan increase in the availability of nutrients to native microbialconsortia which promotes the uptake and metabolization of potentiallyharmful chemicals, thus deterring surface and groundwater contamination.In addition, the recirculation of treated leachate (common in bioreactorlandfills) could act as a multiplier in dosage rates. With enhancedwaste stabilization and improved leachate quality, it is possible forthe thirty years of post-closure care costs to be reduced and the landpromptly reclaimed.

Microbial inoculants and nutrients could be applied using a sprayertruck to either the daily fill cover or directly to incoming waste.Alternate applications include the dosing of leachate ponds and liftstations to improve leachate quality. Continued research onbioaugmentation is warranted to optimize application rate and furtherunderstand the end-products of degradation.

Example 5 Biofilm Formation on HDPE Surface HDPE in Minimal MediaPreparation

Minimal media containing high-density polyethylene (HDPE) as a solecarbon source was augmented with isolated strains of Bacillus subtilisand Bacillus amyloliquefaciens and micronutrients. One preparation wasmade with Treatment-2 including the minimal media, MDG-4954, MDG-2310,micronutrients and HDPE. Inoculation and media replacement occurredweekly over two months (pH=7.0 at 30° C.). Post incubation, an unwashedHDPE sample was sent for SEM imaging to examine the HDPE film surfacefor the presence of Bacillus sp. The HDPE film was removed from thesolution and a few small pieces were cut from the film. The pieces wereallowed to dry and were sputter coated with a thin film ofgold-palladium to facilitate high-resolution SEM. Four separate areaswere examined and micrographs acquired. SEM images of un-washed HDPEfilm show surface cleavage and colonization with rod-shaped particlesabout 1 to 2 μm long consistent with the morphology of Bacillusmicrobes. This implies the initiation of degradation via microbialbiofilm formation (FIG. 4A, FIG. 4B and FIG. 4C).

Example 6 Strain Identification and Uniqueness

The Randomly Amplified Polymorphic DNA PCR method (hereafter referred toas RAPD-PCR) was used to identify genetic variability of each strain.Preparation of the DNA to be used in the RAPD-PCR reaction was done byusing the QIAGEN® Tissue and Blood single column kit (QIAGEN®, Venlo,The Netherlands). FIG. 10 illustrates RAPD-PCR results for strains 2112,8992, 4954, and 2310, with the first and last lanes being a molecularweight ladder. The results show that all four strains are unique fromeach other.

What is claimed is:
 1. A method of treating waste to remove a pollutant,the method comprising contacting the waste with an effective amount ofan isolated Bacillus strain selected from the group consisting ofBacillus strain 8992 (NRRL No. B-67472), Bacillus strain 2112 (NRRL No.B-67473), Bacillus strain 4954 (NRRL No. B-67474), Bacillus strain 2310(NRRL No. B-67471), and combinations thereof, and removing thepollutant.
 2. The method of claim 1 wherein the waste is selected fromthe group consisting of industrial waste, municipal solid waste,landfill waste, soil waste, wastewater, composting waste, contaminatedgroundwater, leachate from waste, polymercontaining waste,hydrocarbon-containing waste, a plastic-containing waste, apolyethylenecontaining waste, a high density polyethylene-containingwaste, and plastic bag-containing waste.
 3. The method of claim 1wherein the pollutant is plastic.
 4. The method of claim 1 wherein thepollutant is a polyethylene.
 5. The method of claim 4 wherein thepollutant is a high density polyethylene.
 6. The method of claim 1wherein the pollutant is an organic compound.
 7. The method of claim 6wherein the organic compound is removed by degradation.
 8. The method ofclaim 1 wherein the pollutant is an inorganic compound.
 9. The method ofclaim 1 wherein at least one of the Bacillus strains has antimicrobialactivity.
 10. The method of claim 9 wherein the antimicrobial activityis against bacteria selected from the group consisting of E. coli,Salmonella, Staphylococcus, Enterococcus, Clostridia, Campylobacter, andcombinations thereof.
 11. The method of claim 1 wherein the Bacillusstrain produces an enzyme selected from the group consisting of an ahydrolase, an oxioreductase, a galactosidase, an NSPase, a phytase, anarabinoxylanase, a cellulase, a hemicelluase, a protease, an amylase, axylanase, an esterase, a lipase, and combinations thereof.
 12. Themethod of claim 1 further comprising treating the waste with anotherbacterial strain selected from the group consisting of another Bacillusstrain, a lactic acid bacterial strain, and combinations thereof. 13.The method of claim 1 wherein the strain is Bacillus strain 8992 (NRRLNo. B-67472).
 14. The method of claim 1 wherein the strain is Bacillusstrain 2112 (NRRL No. B-67473).
 15. The method of claim 1 wherein thestrain is Bacillus strain 2310 (NRRL No. B-67471).
 16. The method ofclaim 1 wherein the strain is Bacillus strain 4954 (NRRL No. B-67474).